The goal of the proposed research is to understand the cellular mechanism whereby glucocorticoids, the primary negative regulators of adrenocorticotrophin (ACTH) secretion, inhibit its release. Ongoing research supports the idea that glucocorticoids cause the rapid (within minutes) induction of a new mRNA and protein in corticotrophs which then acts to suppress ACTH release. The protein mediating these effects needs to be identified and characterized to ultimately understand how and where negative feedback works in corticotrophs. Thus, the major objective of this study is to identify these newly synthesized mRNA(s) and protein(s). Phase I will consist of two steps. First is construction of a cDNA library from AtT20 cells, an immortal murine corticotroph tumor line (already completed). Subtractive hybridization will then be utilized to isolate genes specifically activated by glucocorticoid treatment. Differential expression of these genes will be verified with Northern blots. In phase II, proteins verified to be differentially expressed will be identified in the cDNA library, sequenced, subcloned, expressed in a mammalian expression system, purified and studied in permeabilized AtT20 cells. Permeabilization allows macromolecules to enter the cell while maintaining the integrity of the secretion pathways; this system will allow a bioassay to determine if identified proteins affect ACTH secretion.